scholarly journals Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells.

1980 ◽  
Vol 77 (3) ◽  
pp. 1546-1550 ◽  
Author(s):  
B. K. Davis ◽  
R. Byrne ◽  
K. Bedigian
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipids ◽  
Sperm Cells ◽  
In Vitro Incubation

Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 179-186 ◽  
Author(s):  
S.H. Chen ◽  
Y.H. Yang ◽  
J.P. Liao ◽  
A.X. Kuang ◽  
H.Q. Tian
Keyword(s):  
Plasma Membrane ◽  
Molecular Biology ◽  
Surface Charge ◽  
Enzymatic Digestion ◽  
Egg Cell ◽  
Sperm Cells ◽  
Torenia Fournieri ◽  
Successful Isolation

SummaryEgg cells of Torenia fournieri were isolated from embryo sacs 1 day after anthesis using enzymatic digestion or mechanical dissection. About 5% of the egg cells and zygotes (2–3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added to the mannitol isolation solution, about 18% of the egg cells (8–10 from 50 ovules) could be isolated within 2 h. The egg cells isolated by mechanical dissection could be used for in vitro fertilization studies without any of the potentially deleterious effects of the enzymes on the plasma membrane of egg cell. The egg cells isolated using enzymatic digestion could be used in the study of the molecular biology of female gamete because more egg cells could be isolated with this technique. Using enzymatic digestion, over 10 zygotes from 50 ovules (over 20%) were isolated from the pollinated ovules. Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible in a dicotyledon plant.

scholarly journals Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon

1994 ◽  
Vol 107 (8) ◽  
pp. 2151-2163 ◽  
Author(s):  
B.M. Gadella ◽  
T.W. Gadella ◽  
B. Colenbrander ◽  
L.M. van Golde ◽  
M. Lopes-Cardozo
Keyword(s):  
Plasma Membrane ◽  
Sperm Head ◽  
Breakdown Product ◽  
Prolonged Storage ◽  
Sperm Cells ◽  
Fluorescence Lifetime Imaging ◽  
Outer Leaflet ◽  
Head Surface ◽  
Sperm Plasma Membrane

Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of seminolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of seminolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.

scholarly journals Update on mammalian sperm capacitation: how much does the horse differ from other species?

Reproduction ◽  
2019 ◽  
Vol 157 (5) ◽  
pp. R181-R197 ◽  
Author(s):  
Bart Leemans ◽  
Tom A E Stout ◽  
Catharina De Schauwer ◽  
Sonia Heras ◽  
Hilde Nelis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Sperm Cells ◽  
Cholesterol Depletion ◽  
Sperm Capacitation ◽  
Sperm Plasma Membrane ◽  
Species Specific

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells

2021 ◽  
Vol 118 (49) ◽  
pp. e2025748118
Author(s):  
Lei Wan ◽  
Xi Chen ◽  
Wolfhard Almers
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Membrane Lipids ◽  
Specific Interaction ◽  
Salt Bridges ◽  
Syntaxin 1A ◽  
Dense Cluster ◽  
Low Levels ◽  
Green Fluorescent

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

scholarly journals In vitro evaluation of canine spermatozoa cryopreserved in different extenders

2006 ◽  
Vol 58 (6) ◽  
pp. 1116-1122 ◽  
Author(s):  
E.C.S. Oliveira ◽  
G.C. Juliani ◽  
A.P. Marques Jr. ◽  
M. Henry
Keyword(s):  
Plasma Membrane ◽  
Ethylene Glycol ◽  
Egg Yolk ◽  
Dimethyl Formamide ◽  
Sperm Cells ◽  
Fluorescent Staining ◽  
Progressive Motility ◽  
Functional Integrity ◽  
Hypoosmotic Swelling Test

The efficacy of three extenders, tris-egg yolk-5% ethylene glycol (T1), lactose-egg yolk-5% ethylene glycol (T2) and lactose-egg yolk-5% dimethyl formamide (T3) on preserving the viability of post-thawing canine spermatozoa was evaluated. Three ejaculates per dog were obtained of five animals. The semen was packaged in 0.5ml straws and cooled to 4°C for 120min. The straws were frozen 4cm above the nitrogen level for 15min and thawed in water-bath at 37°C for 60sec and at 75°C for 7sec. Progressive motility and vigour were evaluated immediately after thawing (time 0) and at 30, 60, 90 and 120min. Structural and functional integrity of plasma membrane of the spermatozoa were evaluated, respectively, by fluorescent staining probes and hypoosmotic swelling test. Lactose-egg yolk based extenders showed better cryoprotectant capability and dimethyl formamide was an alternative cryoprotectant agent for dog sperm cells.

scholarly journals Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction. Evidence for a primary capacitation event in boar spermatozoa

1995 ◽  
Vol 108 (3) ◽  
pp. 935-946 ◽  
Author(s):  
B.M. Gadella ◽  
M. Lopes-Cardozo ◽  
L.M. van Golde ◽  
B. Colenbrander ◽  
T.W. Gadella
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Phase Segregation ◽  
Sperm Head ◽  
Lipid Phase ◽  
Sperm Cells ◽  
Fluorescence Lifetime Imaging ◽  
Boar Spermatozoa

In order to extend the static information of immunolabelling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3-sulphate)-beta 1–1′[(N-lissamine rhodaminyl)-12-aminodode-canoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribution over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejaculated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-beta 1–1′[(N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatorial segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe distribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incorporated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.

scholarly journals Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

2012 ◽  
Vol 23 (18) ◽  
pp. 3743-3753 ◽  
Author(s):  
Cao Guo Yu ◽  
Tony J. C. Harris
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Membrane Lipids ◽  
Cell Types ◽  
Molecular Networks ◽  
Pdz Domains ◽  
In Vitro Characterization ◽  
Wide Range ◽  
And Function

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D—enzymes that produce phosphatidic acid—reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

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